Over millions of years under severe environmental pressure in murky waters, the cyanobacterial light-harvesting phycobiliprotein allophycocyanin (APC) evolved for excitation and emission maxima in the near-infrared (NIR) optical window (650-900 nm). Harnessing the power of this natural selection to develop a new generation of bright and robust fluorescent proteins (FPs) for a range of applications, including deep imaging of mammalian tissue, is needed. (Miyawaki, A. Nature Methods, 13(9):729-730, 2016).
While a variety of fluorescent proteins are known, including: eGFP, mCherry, mCardinal, IFP1.4, IFP2.0, and iRFP713, including work by Atsushi Miyawaki's lab. Traditional FUCCI uses a green and red fluorescent proteins evolved from jellyfish and coral. Despite this, there remains a need in the art for additional proteins with increased stability and increased emission maxima ranges for use in detection methods, including deep tissue imaging.
The present invention provides small ultra-red fluorescent proteins (smURFPs) or smURFP variant polypeptides which are biophysically the brightest far-red and near-infrared fluorescent proteins, fill a spectral gap in excitation wavelength, express efficiently with minimal toxicity, and do not produce hydrogen peroxide. In addition, unlike its precursor, TeAPCα, smURFP variant polypeptides do not require a lyase to covalently attach its chromophore.
The present invention provides novel far-red/near-infrared fluorescent proteins from allophycocyanin α-subunt from cyanobacteria Trichodesmium erthraeum, referred to small ultra-red fluorescent proteins (smURFPs). These proteins are in the spectral space 600-650 nm, a space not covered by commercially available reagents. smURFPs contain an enlarged chromophore binding site amenable for tagging to different probes using click chemistry. Moreover, biliverdin tagged proteins are suitable for in vivo imaging in cancer and other maladies where hydrogen peroxide is generated. Thus the smURFPs of the invention meet an unmet need.